ELUCIDATING THE ROLES OF WNT/BETA-CATENIN SIGNALING IN LIVER DISEASE, CANCER, AND REGENERATION

Owing to its strategic location in the body along with performing over 500 daily functions, liver health is indispensable to survival. Following any form of acute or chronic hepatic injury, a repair process is induced which, if successful, restores histology and function. However, continuous insult can lead to unremitting immune response, hepatocyte death, proliferation, wound healing, and sets the stage for DNA damage and errors, eventually leading to dysplasia and neoplasia. Wnt/β-catenin signaling is implicated in a variety of processes to maintain liver homeostasis and promote regeneration, but its dysregulation is often evident in chronic liver diseases and cancer. To further understand the role and regulation of the Wnt/β-catenin pathway in hepatic pathophysiology, we focused on studying this pathway in a model of chronic iron overload replicating hereditary hemochromatosis (HH), hepatocellular carcinoma (HCC), a primary liver tumor with rising incidence, and in metabolic zonation and liver regeneration (LR) after partial hepatectomy (PH), two fundamental attributes innate to the liver. The underlying goal of our studies was to address the role of β-catenin in hepatocyte biology, and identify the cell source and identity of Wnt ligands that regulate β-catenin signaling in hepatocytes, both in hepatic health and in disease. HH is a genetic iron overload disorder with no cure, invasive treatments, and no representative animal models that recapitulate human disease. β-Catenin knockout mice (bKO), when subjected to chronic iron overload led to remarkable hepatic injury compared to control mice (bCON) and displayed inflammation, steatosis and fibrosis due to enhanced oxidative stress and lipid peroxidation. This led us to identify an important role of β-catenin in regulating the redox state of hepatocytes as we alleviated the pathology by supplementing the antioxidant N-acetyl-L-cysteine to bKO while challenging them with iron overload. Additionally, bKO on high iron diet serve as a novel animal model that robustly mimic disease development and progression of an HH patient. Chronic injury like HH often progresses to HCC accompanied by ongoing hepatocyte death and proliferation, along with infiltration of immune cells including macrophages. We next assessed if hepatocytes and macrophages could be the source of Wnt ligands that may activate non-mutated β-catenin in hepatocytes to contribute to injury, inflammation and HCC. We generated hepatocyte-specific (HP-KO) and macrophage-specific (MP-KO) Wntless knockout mice, unable to secrete Wnts from respective cell types and subjected them to diethylnitrosamine/carbon tetrachloride (DEN/CCl4). This model yields non-β-catenin mutated HCC with 100% penetrance by five months. HP-KO and respective controls (HP-CON) had comparable tumor burden albeit with some distinct signaling indicating overall that hepatocytes are not a major contributor of Wnts in HCC in this model. MP-KO displayed two distinct, litter-specific phenotypes. In one group, MP-KO had greater tumor burden than control mice (MP-CON), and in another group MP-KO had reduced tumor burden compared to MP-CON. We revealed different HCC phenotypes in MP-CON from both groups, suggesting MP Wnts will promote or suppress tumors depending on the underlying oncogenic and inflammatory milieu. Finally, we queried the role of cell-specific Wnts in metabolic zonation and LR. Using endothelial cell-specific Wntless knockout mice (EC-KO), we confirmed EC to be the source of Wnt ligands that directed β-catenin activation in pericentral hepatocytes. Also, EC lining sinusoids regulated Cyclin D1 expression and eventually hepatocyte proliferation during LR after PH. We next identified Wnt2 and Wnt9b as the Wnts chiefly from EC to be regulators of hepatic zonation and during LR after PH. Finally, we provide in vitro evidence that shear stress produced after PH may initiate Wnt expression in EC and hence may initiate the process of LR. Overall, our data elucidates the role and regulation of Wnt/β-catenin signaling and begins to outline the complex paracrine cell-molecule circuitry of this intricate pathway in hepatic health and disease

Dietary carbohydrates and breast cancer risk: A prospective study of the roles of overall glycemic index and glycemic load

We examined breast cancer risk in association with overall glycemic index (GI), glycemic load (GL), and dietary carbohydrate and sugar intake in a prospective cohort of 49,613 Canadian women enrolled in the National Breast Screening Study who completed a self‐administered food frequency questionnaire between 1980 and 1985. Linkages to national mortality and cancer databases yielded data on deaths and cancer incidence, with follow‐up ending between 1998 and 2000. During a mean follow‐up of 16.6 years, we observed 1,461 incident breast cancer cases. GI, GL, total carbohydrate and total sugar intake were not associated with breast cancer risk in the total cohort. However, there was evidence of effect modification of the association between GI and breast cancer risk by menopausal status (p = 0.01), the hazard ratio for the highest versus the lowest quintile level of GI being 0.78 (95% CI = 0.52–1.16; ptrend = 0.12) in premenopausal women and 1.87 (95% CI = 1.18–2.97; ptrend = 0.01) in postmenopausal women. The associations between GI and GL were not modified by body mass index (BMI) or by vigorous physical activity among pre‐ or postmenopausal women. Similarly, the associations between GI/GL and risk in postmenopausal women were not modified by BMI, vigorous physical activity, or ever use of hormone replacement therapy (HRT), although the associations were slightly stronger among those who reported no vigorous physical activity (ptrend = 0.02), among those who reported ever using HRT (ptrend = 0.02) and among normal‐weight women (BMI \u3c 25 kg/m2; ptrend = 0.03). Our data suggest that consumption of diets with high GI values may be associated with increased risk of breast cancer among postmenopausal women, possibly more so among subgroups defined by participation in vigorous physical activity, ever use of HRT and those who are not overweight

State of the art in silico tools for the study of signaling pathways in cancer

In the last several years, researchers have exhibited an intense interest in the evolutionarily conserved signaling pathways that have crucial roles during embryonic development. Interestingly, the malfunctioning of these signaling pathways leads to several human diseases, including cancer. The chemical and biophysical events that occur during cellular signaling, as well as the number of interactions within a signaling pathway, make these systems complex to study. In silico resources are tools used to aid the understanding of cellular signaling pathways. Systems approaches have provided a deeper knowledge of diverse biochemical processes, including individual metabolic pathways, signaling networks and genome-scale metabolic networks. In the future, these tools will be enormously valuable, if they continue to be developed in parallel with growing biological knowledge. In this study, an overview of the bioinformatics resources that are currently available for the analysis of biological networks is provided

Circulating microRNAs in sera correlate with soluble biomarkers of immune activation but do not predict mortality in ART treated individuals with HIV-1 infection: A case control study

Introduction: The use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals. Materials and Methods: A set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed. Results: None of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR- 145 correlated with nadir CD4+ T cell count. Discussion: No associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Mice lacking liver-specific β-catenin develop steatohepatitis and fibrosis after iron overload

Background & aimsIron overload disorders such as hereditary hemochromatosis and iron loading anemias are a common cause of morbidity from liver diseases and increase risk of hepatic fibrosis and hepatocellular carcinoma (HCC). Treatment options for iron-induced damage are limited, partly because there is lack of animal models of human disease. Therefore, we investigated the effect of iron overload in liver-specific β-catenin knockout mice (KO), which are susceptible to injury, fibrosis and tumorigenesis following chemical carcinogen exposure.MethodsIron overload diet was administered to KO and littermate control (CON) mice for various times. To ameliorate an oxidant-mediated component of tissue injury, N-Acetyl-L-(+)-cysteine (NAC) was added to drinking water of mice on iron overload diet.ResultsKO on iron diet (KO +Fe) exhibited remarkable inflammation, followed by steatosis, oxidative stress, fibrosis, regenerating nodules and occurrence of occasional HCC. Increased injury in KO +Fe was associated with activated protein kinase B (AKT), ERK, and NF-κB, along with reappearance of β-catenin and target gene Cyp2e1, which promoted lipid peroxidation and hepatic damage. Addition of NAC to drinking water protected KO +Fe from hepatic steatosis, injury and fibrosis, and prevented activation of AKT, ERK, NF-κB and reappearance of β-catenin.ConclusionsThe absence of hepatic β-catenin predisposes mice to hepatic injury and fibrosis following iron overload, which was reminiscent of hemochromatosis and associated with enhanced steatohepatitis and fibrosis. Disease progression was notably alleviated by antioxidant therapy, which supports its chemopreventive role in the management of chronic iron overload disorders.Lay summaryLack of animal models for iron overload disorders makes it hard to study the disease process for improving therapies. Feeding high iron diet to mice that lack the β-catenin gene in liver cells led to increased inflammation followed by fat accumulation, cell death and wound healing that mimicked human disease. Administration of an antioxidant prevented hepatic injury in this model